Penicillin began the antibiotic revolution. Providing the first real weapon against microbial infections, penicillin (see FIG. 1) first appeared to be a xe2x80x9cmagic bulletxe2x80x9d that would cure all of man""s ills. Infectious microbes soon developed resistance to penicillins, however. Great efforts in the pharmaceutical industry have focussed and still focus on the development of alternative antibiotics. One of the most useful families of agents is the cephalosporins (see FIG. 2).
The first cephalosporin, cephalosporin C, was isolated from Cephalosporium acremonium (also known as Acremonium chrysogenum) in 1954. C. acremonium produces cephalosporin C by first synthesizing penicillin N, and then converting this penicillin into cephalosporin C according to the pathway presented in FIG. 3. As shown in FIG. 3, penicillin N is first converted to deacetoxycephalosporin C (DAOC) through oxidative expansion catalyzed by an enzyme known as xe2x80x9cDAOC synthasexe2x80x9d (DAOCS), or xe2x80x9cexpandasexe2x80x9d. A hydroxylase activity, which in C. acremonium is part of the same DAOCS enzyme, then converts the DAOC to deacetylcephalosporin C (DAC). In the final step of the conversion, an acetyl transferase substitutes an acetoxy group for the DAC hydroxyl and thereby produces cephalosporin C.
Further study revealed that C. acremonium is not the only organism that produces cephalosporins from penicillin N. In particular, S. clavuligerus also has both expandase and hydroxylase activities, which activities are separable from one another in this organism. Unfortunately, however, no organism has been identified that naturally produces any commercially useful cephalosporin. Commercially useful cephalosporins (see, for example, FIG. 2B) are typically produced by chemical ring expansion of, for example, penicillin G to yield deacetoxycephalosporin G. Other cephalosporins can then be produced through enzymatic removal of the deacetoxycephalosporin G side chain (phenylacetyl) and substitution of a different side chain. The multi-step chemical ring expansion process is time consuming, expensive, and polluting.
Alternatively, commercially useful cephalosporins could be produced by isolating either the DAOC or the DAC intermediate from C. acremonium or S. clavuligerus fermentations, and chemically treating the isolate to eliminate the D-xcex1-aminoadipyl side chain and produce a substrate (7-aminodeacetoxycephalosporanic acid [7-ADCA] or 7-aminodeacetylcephalosporanic acid [7-ADAC]) that can subsequently be chemically treated to generate a medically useful cephalosporin (see FIG. 4). Although it avoids the chemical ring expansion step, this strategy is also expensive, since the levels of DAOC or DAC that naturally accumulate are small. There is a need for an improved system for producing cephalosporins.
In particular, there is a need to develop a system that allows cephalosporin production from a penicillin other than penicillin N. Preferably, the system would allow cephalosporin production from an inexpensive penicillin such as penicillin G or penicillin V. As shown in FIG. 5, penicillin G conversion would produce intermediates (deacetoxycephalosporin G [DAOG], deacetylcephalosporin G [DAG]) that could be treated with penicillin acylase to produce the same 7-ADCA or 7-ADAC substrates mentioned above.
Various efforts have been made to utilize the C. acremonium or S. clavuligerus expandase enzyme either alone or with a hydroxylase enzyme to convert penicillins other than penicillin N into a cephalosporin or cephalosporin intermediate or substrate. Such efforts have almost uniformly failed. Many researchers have reported that the C. acremonium and S. clavuligerus expandase enzymes have very narrow specificity and fails to expand penicillins other than penicillin N and certain very close relatives.
For example, Kohsaka and Demain, the original discoverers of C. acremonium expandase, have reported that only penicillin N, and not penicillin G or 6-aminopenicillanic acid (6-APA), are substrates for expandase activity in crude extracts (Kohsaka et al., Biochem. Biophys. Res. Commun. 70(2):1976:465-473, 1976; Demain et al., U.S. Pat. No. 4,178,210, issued Dec. 11, 1979). Further work by this group has demonstrated that partially purified enzyme does not expand adipyl-6-APA, ampicillin, or penicillin G (Kupka et al., FEMS Microbiol. Lett. 16:1-6, 1983).
Similarly, researchers have reported that the S. clavuligerus expandase expands the ring of penicillin N, but not that of at least twenty other penicillins, including penicillin G, penicillin V, penicillin K, penicillin dihydroF, adipyl-6-APA, m-carboxyphenylacetyl-6-APA, ampicillin, butyryl-6-APA, D-glutamyl-6-APA, and ampicillin (Jensen et al., J. Antibiot. 35:1351-1360, 1982; Dotzlaf et al., J. Biol. Chem. 264:10219-10227, 1989; Yeh et al. in 50 Years of Penicillin: History and Trends [Kleinkauf et al., eds.], Public, Prague, pp. 208-223, 1994; Maeda et al., Enzyme Microb. Technol. 17:231-234, 1995).
One group has reported that Penicillium chrysogenum cells that have been engineered to express the S. clavuligerus expandase gene can produce adipyl-7-aminodeacetoxycephalosporanic acid (adipyl-7-ADCA) when grown in the presence of adipic acid (Conder et al., U.S. Pat. No. 5,318,896, issued Jun. 7, 1995; Crawford et al., Bio/Technol. 13:58-62, 1995). P. chrysogenum cells are capable of converting adipic acid to adipyl-6-APA; the observation of adipyl-7-ADCA production by the recombinant cells therefore suggests that the S. clavuligerus expandase, when expressed in P. chrysogenum cells, may be able to expand the endogenous adipyl-6-APA.
A small number of other studies have reported some ability of S. clavuligerus or C. acremonium expandase enzymes to expand D-carboxymethylcysteinyl-6-APA, a very close relative to penicillin N (Bowers et al., Biochem. Biophys. Res. Commun. 120:607-614, 1984) and adipyl-6-APA (Baldwin et al., J. Chem. Soc. Chem. Commun. 1466:374-375, 1987; Shibata et al., Bioorg. Med. Chem. Lett. 6:1579-1584, 1996), in vitro. One group (Baldwin et al., J. Chem. Soc. Chem. Commun. 1466:374-375, 1987) has also suggested that m-carboxyphenylacetyl-6-APA, D-glutamyl-6-APA, and glutamyl-6-APA might also serve as in vitro substrates, albeit at very low levels. Subsequent work failed to confirm these reports, however (Yeh et al., in 50 Years of Penicillin: History and Trends [Kleinkauf et al, eds], Public, Prague, pp. 208-223, 1994).
One brief abstract reported that a recombinant form of S. clavuligerus expandase, when expressed in and purified from Escherichia coli, might be able to expand penicillin G (Baldwin et al., Abstract P-262, Abstracts of the 7th International Symposium on Genetics of Industrial Microorganisms, Montreal, Jun. 26-Jul. 1, 1994, pg. 184). Unfortunately, the report did not contain sufficient detail to allow ready duplication of the results and no subsequent work has confirmed the finding.
Thus, the prior art attempts to develop an improved system for producing cephalosporins from penicillins other than penicillin N have generally failed. In particular, efforts to develop a system that utilizes penicillin G as a substrate have been unsuccessful. There remains a need for development of improved systems for converting penicillins other than penicillin N. Particularly desirable systems would utilize exogenously-added penicillins rather than relying on in vivo microbial penicillin production. Particularly preferred systems would obviate the need for multi-step chemical ring expansion methods.
The present invention provides techniques and reagents for the bioconversion of penicillins other than penicillin N into cephalosporins or cephalosporin precursors. The inventive conversion system allows biological ring expansion of penicillin substrates such as penicillin G, and replaces the multi-step chemical ring expansion process currently performed in industry. The inventive system can utilize growing or resting cells (free or immobilized), or isolated expandase (crude or purified), and is capable of converting exogenously-added penicillins. The inventive system can be applied to any penicillin substrate, including natural penicillins (e.g., penicillin G), biosynthetic penicillins (e.g., penicillin V), semisynthetic penicillins (e.g., ampicillin), and/or synthetic penicillins.
xe2x80x9cCephalosporin precursorxe2x80x9dxe2x80x94The term xe2x80x9ccephalosporin precursorxe2x80x9d, as used herein, refers to a compound that, through one or more chemical reactions not relying on an expandase, can be converted into a cephalosporin. This term is intended to encompass many compounds that are also cephalosporins, so long as they are convertible into other cephalosporins. Preferred cephalosporin precursors have the structure depicted in FIG. 2A, and include 7-ADCA, 7-ADAC, 7-ACA, DAOG, DAG, cephalosporin G, and cephamycin G. DAOG and DAG are particularly preferred.
xe2x80x9cExogenous substratexe2x80x9dxe2x80x94The term xe2x80x9cexogenous substratexe2x80x9d, as used herein, refers to a substrate that is added to a reaction and is not produced internally by a cell producing expandase. That is, when the expansion reaction occurs inside a cell that produces both the expandase and the penicillin substrate on which the expandase acts, that substrate is an xe2x80x9cendogenousxe2x80x9d substrate. By contrast, if the penicillin substrate is added e.g., to cells producing the expandase, that substrate is exogenous, even if it is the same chemical compound that is being (or could be) produced by the cell.
xe2x80x9cIsolatedxe2x80x9dxe2x80x94The term xe2x80x9cisolatedxe2x80x9d, when applied to a compound that exists in nature, means (i) separated from at least some of the components with which it is normally associated in nature; and/or (ii) produced or prepared through a process (e.g., involving in vitro synthetic chemistry) that does not occur in nature.
xe2x80x9cPurifiedxe2x80x9dxe2x80x94A compound is considered xe2x80x9cpurifiedxe2x80x9d when it is at least about 50% pure, preferably at least 70-80% pure, more preferably at least about 90% pure, yet more preferably at least 95% pure, and most preferably at least 99% pure.
xe2x80x9cRecombinantxe2x80x9dxe2x80x94The term xe2x80x9crecombinantxe2x80x9d, as used herein, means produced through a method relying on techniques of recombinant DNA technology. For example, an expandase gene is separated from DNA with which is normally associated in nature and is introduced into an expression vector, the gene in the context of the vector is a xe2x80x9crecombinantxe2x80x9d gene. Similarly, the protein expressed from the gene is a xe2x80x9crecombinantxe2x80x9d protein.